Preparation And Investigation On Performance Of Phosphoprotein Enrichment Temperature-responsive Chromatographic Materials

Chromatographic stationary phases with specific capturing phosphoproteins play an extremely important role in biological sample pretreatment.However,when the captured protein is released,it is generally required to change the pH of the mobile phase or to use an eluent.The mobile phase and eluent used are often high concentrations,salt solutions with extreme pH or toxic organic reagents.These reagents destroy the activity and structure of phosphorylated proteins easily,resulting in the loss of their vital biological information.In addition,the mobile phase after switching the column takes longer to restore the balance,reducing the experimental efficiency.In order to solve the above problems,we introduce temperature-sensitive materials into the chromatographic stationary phase to achieve the enrichment and release of phosphorylated proteins by changing the temperature,in which we use water as the mobile phase.This approach overcomes the shortcomings of traditional methods,and makes the separation process safer and simplerIn this study,based on the reversible addition-fragmentation chain transfer polymerization（RAFT）method,we prepared three kinds of thermally responsive materials,concluding silica@pNIPAAm-nanoTiO₂、silica@p（NIPAAm-co-AMPA）-Ti⁴⁺and silica@p（NIPAAm-co-IPPA）-Ti⁴⁺,by using NIPAAm with temperature sensitivity and nano-TiO₂/Ti⁴⁺with high specific binding capacity to phosphorylation protein.Besides,the three materials were successfully applied to the enrichment and separation of small molecule phosphate compounds and phosphopeptides.Specific research includes the following aspects:1.Based on the SI-RAFT method,silica@pNIPAAm-nanoTiO₂,a kind of Metal Oxide Affinity Chromatography,was synthesized by the rapid introduction of functional groups.The synthesis of the material was confirmed by infrared and X-ray photoelectron spectroscopy.The grafting rate and the lowest critical temperature were measured by TG and DSC.The results showed that the material had qualified temperature-sensitive properties.The grafting conformation and mobile phase pH of the material were optimized before testing the properties and found that when the material grafting ratio was 10%-15%,the graft density was 30%,and the mobile phase pH was 6,it had the best seperate effect.Finally,the material successfully achieved the capture and release of adenosine triphosphate and casein phosphopeptides.2.Based on the SI-RAFT method,silica@p（NIPAAm-co-AMPA）-Ti⁴⁺,a kind of Immobilized Metal Ion Affinity Chromatography material,was synthesized by the rapid introduction of functional groups.The synthesis of the material was confirmed by FTIR and X-ray photoelectron spectroscopy.The grafting ratio and the LCST of the material were measured by TGA and DSC respectively.The results showed that the material has excellent temperature-sensitive properties.The grafting conformation and mobile phase pH of the material were optimized before use.It was found that when the grafting ratio is 20%-25%,the graft density is 50% and the mobile phase pH is 7,the material has the best seperate effect.Finally,the material achieved the capture and release of adenosine triphosphate and casein phosphopeptides successfully.By the way,service life of the material could be extended by the supplement Ti⁴⁺online.3.Based on the SI-RAFT method,silica@p（NIPAAm-co-IPPA）-Ti⁴⁺,a kind of Immobilized Metal Ion Affinity Chromatography material,was synthesized by one-step polymerization.The synthesis of the material was confirmed by FTIR and X-ray photoelectron spectroscopy.Scanning electron microscopy（SEM）proved that the synthetic route can effectively protect the morphology of silica.The ratio of IPPA and NIPAAm was optimized and found that IPPA achieved the best capture and release when it accounted for 15% of the total number of moles.Finally,the material successfully achieved multiple enrichment and separation of casein phosphopeptides.And it achieved the separation of phosphorylated proteins from bio-sample.